1. Field of the Invention
The present invention relates to an apparatus and method for growing a biological macromolecular crystal such as protein, and an apparatus and method for characterization of a biological macromolecular crystal.
2. Description of the Related Art
In this specification, a biological macromolecule means a crystallizable macromolecular substance such as protein, nucleic acid, enzyme and an antibody.
A biological macromolecule has various roles of chemical reaction for life support in a living body. To elucidate a mechanism of vital activity, or to develop a medicine of a high effect, it is very important to understand functions of a biological macromolecule. The functions of a biological macromolecule is deeply associated with a three-dimensional structure of the biological macromolecule. In order to understand the functions of the biological macromolecule, various methods for elucidating the three-dimensional structure have been attempted.
Among them, X-ray structure analysis is one of the most effective methods for investigating the three-dimensional structure.
Performing of the X-ray structure analysis needs preparation of a biological macromolecular crystal. Generally, it is difficult to predict a condition for crystallization, and it is also necessary to experimentally screen many parameters such as a crystallizing agent type, crystallizing agent concentration, biological macromolecular concentration, buffer type, pH and temperature. Usually, crystallization takes several days to several weeks, so that it takes a large amount of labor and time to grow a single high-quality crystal. Therefore, a step of obtaining a single high-quality crystal of the biological macromolecule is a bottleneck for the X-ray structure analysis.
Recently, biological macromolecule crystallization using a space environment has been carried out. In a space environment, since gravity does not act on the crystal nucleus, the crystal nucleus does not sink to a bottom part of solution, and no convection of air around the crystal can be realized. Accordingly, it is considered that space can provide good environment for crystal growth. For this reason, it has been attempted to produce high-quality crystal in space, and to recover the crystal for carrying out the X-ray structure analysis on the earth.
Biological macromolecule crystallizing means and biological macromolecule X-ray structure analysis are disclosed in Documents 1 through 13.    [Document 1] Biological Crystal Producing Handbook (publisher: Maruzen Inc., author: Reimei Hirayama)
[Document 2] X-ray analysis of protein (publisher: Kyouritu Inc. author: Mamoru Sato)    [Document 3] Japanese Laid-Open Patent Publication No. 2001-213699    [Document 4] Japanese Laid-Open Patent Publication No. 2002-233702    [Document 5] Japanese Laid-Open Patent Publication No. 6-300718    [Document 6] Japanese Laid-Open Patent Publication No. 11-94773    [Document 7] Japanese Patent No. 2650274    [Document 8] Japanese Laid-Open Patent Publication No. 6-62848    [Document 9] Japanese Laid-Open Patent Publication No. 6-321700    [Document 10] Japanese Laid-Open Patent Publication No. 6-183400    [Document 11] Japanese Laid-Open Patent Publication No. 6-157598    [Document 12] Japanese Laid-Open Patent Publication No. 6-116098    [Document 13] Japanese Laid-Open Patent Publication No. 5-25000
FIGS. 1A and 1B show a principle of “crystallization of protein by a vapor diffusion method” disclosed in Document 2.
In FIG. 1B, concentration change of biological macromolecules (protein) in crystallizing solution is schematically shown with respect to concentration change of a crystallizing agent (salt) in the solution. In this drawing, “(1)” designates a solubility curve, and “A” designates an unsaturated region A where a biological macromolecule is completely dissolved. “B”, “C” and “D” designate oversaturated regions B, C and D where association of the biological macromolecules occurs. However, stable nucleation requires a certain level of oversaturation. Accordingly, in the low-level oversaturated region B, even if the association occurs, the association is unstable, so that the macromolecules are disassociated in a short time. On the other hand, in the high-level oversaturated region D, even if the association occurs, deposit is generated without occurring of specific interaction necessary for the nucleation. Therefore, for biological macromolecule crystallization, it is necessary to adjust the solubility of the biological macromolecule such that the solution is brought into the region C.
As shown in FIG. 1A, in the vapor diffusion method, there are three methods of placing the biological macromolecular solution, that is, hanging drop method, sitting drop method and sandwich method. In the vapor diffusion method, a droplet of biological macromolecular (protein) solution containing a crystallizing agent (precipitating agent) is placed in a closed container in which buffer solution containing a precipitating agent is also placed. In this container, the biological macromolecules are crystallized with the biological macromolecular solution droplet being vaporized. At first, the biological macromolecular solution containing the crystallizing agent stays in the unsaturated region A, the biological macromolecules are completely dissolved. After water in the biological macromolecular solution gradually evaporates in the closed container, the state of the biological macromolecular solution is changed to the oversaturated region C. The crystal is precipitated in the region C, and then, the concentration of the biological macromolecular solution gradually decreases, so that the state of the biological macromolecular solution reaches the solubility curve (1) where the crystal precipitation stops.
Document 3 of which title is “Crystal Adjusting Device, Crystal Adjusting Method and Device Kit” discloses a screening apparatus that aims to make conditions suitable to crystallization of various macromolecules in a short time.
According to Document 3, as shown in FIGS. 2A and 2B, the crystal adjusting apparatus 170 includes a first board 171 and a plurality of second boards 172. The first board 171 has a plurality of penetration holes separated from each other. The second boards 172 each have plural surface portions having different surface potentials or different zeta potentials. The second boards 172 are arranged to cover a plurality of the penetration holes 173. At each of a plurality of portions 174 for holding solution, the plural surface portions that have different surface potentials or zeta potentials contact with the solution. In FIG. 2B, the reference numeral 175 designates a protrusion part, and 176 a concave part.
Document 4 of which title is “Crystal Growing Apparatus, Apparatus Components and Crystal Growing Method” discloses an apparatus that aims to control a water vaporizing speed without changing a type or concentration of a precipitating agent in a process of crystallizing biological macromolecules by the vapor diffusion method.
According to Document 4, as shown in FIG. 3, the crystal growing apparatus includes a closable container 211 that receives several droplets of biological macromolecular solution, and a separation plate 213 that separates an inside space of the container 211 into a first room 218 and a second room 219. The first room 218 receives a precipitating agent 220, and the second room 219 receives the droplets 221. In a crystallization process, a substance diffuses between the first and second rooms through penetration holes 232 of the separation board 213. In FIG. 3, the reference numeral 212 designates a protrusion part, 214 an upper wall part, 215 a cover, and 231 a concave part.
Document 5 of which title is “X-ray Analyzing Apparatus” discloses an apparatus that aims to easily perform X-ray analysis of a minute region of a sample by greatly changing a position of the sample.
According to Document 5, as shown in FIG. 4, an X-ray optical element that has focusing and imaging functions and has a long operating distance is incorporated in an X-ray generation apparatus. A sample scanning table 310 is provided with a goniostage 315, a rotary stage 313, a straight moving stages 317, 318 and 319 that can precisely move in directions of three axes, respectively. The sample scanning table 310 is installed such the rotational axis of the sample scanning table 310 is included in the focal plane of the X-ray optical element. Further, a rotatable X-ray position detector or an X-ray energy detector is provided to move around the sample scanning table 310. In FIG. 4, the reference numeral 302 designates an X-ray optical axis, and 320 a large sample holder.
Document 6 discloses an X-ray analyzing apparatus that aims to efficiently measure many samples with high accuracy.
According to the X-ray analyzing apparatus 401 of Document 6 shown in FIG. 5, when a plurality of samples are set in a sample magazine 409, a compressed spring provided in the sample magazine 409 pushes the sample via a plate member toward a body part of a jig 408 so that the pushed sample can enter a sample hole 413a or 413b of an arm member 412 facing the sample magazine 409 and be pushed against a front edge surface of an upper arm part and a step of a lower arm part of the jig 408 that extends out from the sample hole. In this state, an X-ray generator 402 emits an X ray 405 to the sample, and the X ray 405 reflected and diffracted by the sample is detected by an X-ray detector 403. Next, a driving motor 416 moves the arm member 412 to a position where the other sample hole 413a or 413b faces the sample magazine 409, and the sample in the sample magazine 409 is made to enter the other sample hole 413a or 413b while the measured sample is made to drop to a receiving plate 418a or 418b. Then, next measurement is performed. In FIG. 5, the reference numeral 407 designates a rotary table.
The above-described vapor diffusion method is the most used for crystallizing the biological macromolecules. In the vapor diffusion method, a droplet of solution that is a mixture of biological macromolecular solution and crystallizing agent solution is placed in a closed system in which another crystallizing agent solution having higher concentration of a crystallizing agent than that of the droplet is also placed. In this manner, when the system reaches vapor equilibrium of vapor pressure between the droplet and the crystallizing agent solution, the droplet is concentrated so that the concentration of the crystallizing agent (and also the concentration of the biological macromolecules) can be increased.
FIG. 6 is a schematic illustration of the hanging drop method that is the most used manner in the vapor diffusion method. The hanging drop method uses a plate having a plurality of wells that function as biological macromolecule crystal growing rooms to perform crystallization. For example, a commercially available plate has four columns and six rows, that is, 24 wells.
Except that a preferable condition for crystal precipitating and growing is previously known, it is rare to obtain a high-quality crystal of biological macromolecules in the droplet. It is difficult to theoretically predict a preferable condition for the crystallization of the biological macromolecules. Generally, crystallization is attempted by changing, little by little, a condition such as a crystallizing agent type, crystallizing agent concentration, a biological macromolecular concentration, a type of buffer solution dissolving the biological macromolecules, pH, and a crystallizing temperature. Such a screening process of trial and error is repeatedly performed. Generally, after attempting many conditions, a good condition can be found for crystallization. Accordingly, it is desired to develop a crystallizing method of efficiently screening crystallizing conditions as easily as possible.
A conventional vapor diffusion method such as the hanging drop method has an advantage of a small necessary amount of biological macromolecular solution. However, a combination of a droplet (including biological macromolecular solution) and crystallizing agent solution needs to be set in each well of a closed system. Accordingly, there is a large trouble of setting droplets in many wells.
Change of a concentration of a droplet in the conventional vapor diffusion method shown in FIG. 6 is as follows.
(1) After setting, as time elapses, the droplet is concentrated by water vaporization, and the concentration of the biological macromolecules and the concentration of the crystallizing agent are increased.
(2) The concentration of the biological macromolecules and the concentration of the crystallizing agent are changed to a region where a nucleus can be formed.
(3) Subsequently, crystal growth occurs, and as protein molecule in the solution is attracted to the crystal, the concentration of the protein is decreased.
Through the above stages (1) to (4), the concentration of the crystallizing agent in the droplet is increased toward the concentration of the crystallizing agent solution. A concentrating speed depends on a size and shape of a container (closed system) as well as a temperature, a droplet size, a crystallizing agent type, and a crystallizing agent concentration. Since a droplet concentration changing speed affects kinetics of crystal growth, a droplet concentration changing speed is considered to be one of condition parameters for obtaining high-quality crystal. Conventionally, a condition for a concentration changing speed is not actively changed except for changing of a temperature, a droplet size, a crystallizing agent type, and a concentration. Accordingly, there was a possibility that an important condition was not screened.
To elucidate functions of a biological macromolecule, it is inevitable to know a three-dimensional structure of the biological macromolecule. For this reason, X-ray diffraction measurement for crystal structure analysis is performed on a single crystal of biological macromolecules obtained by crystallization. The produced biological macromolecular crystal is taken out from an apparatus, and is mounted on a jig to be attached to a goniometer head of an X-ray diffraction measuring apparatus. Crystallization is performed in various methods, but the produced crystal is generally mounted on the jig in the following method.
FIG. 7A shows one crystal mounting method that uses a capillary. A crystallizing mother liquid is put in a thin glass-made capillary having a thickness of about 1 mm, and both ends of the capillary are sealed. Then, the capillary is attached to a goniometer. FIG. 7B shows another crystal mounting method that uses a cryoloop. A cryoloop that has a diameter of about 0.1 mm to 1 mm and that is made of nylon fiber having a diameter of 10 μm to 20 μm is commercially available. The mother liquid is scooped together with a crystal by the loop so that the crystal can be held by surface tension. This method is used mainly for X-ray diffraction at a low temperature by streaming extremely-low-temperature gas such as nitrogen onto the crystal held by the loop. Thus, one target crystal is taken out from an apparatus, and is mounted on a some sort of jig to be attached to a goniometer.
Structure analysis of a biological macromolecular becomes more important, and researchers in several countries are performing intense study on this. However, among a large number of biological macromolecule types that function in a living body, a three-dimensional structure of only a small part of them became clear. It is required to raise a processing speed of the crystal structure analysis. For this, it is desired to achieve high throughput by automation or efficiency improvement of manual working.
One obstacle for the high throughput is work of taking out a produced crystal, mounting the crystal on a jig, and attaching the jig to a goniometer. Since this work is performed for each X-ray diffraction, a great trouble is required. Furthermore, this work requires a high skill, and automation of this work is difficult. Accordingly, for high throughput, it is desired to efficiently perform the work of taking out a crystal, mounting the crystal on a jig, and attaching the jig to a goniometer.